The Diels-Alder reaction is a cycloaddition reaction between a conjugated diene and a substituted alkene or alkyne (e.g., a dienophile), to form a substituted cyclohexene system. The reaction forms two carbon-carbon bonds and up to four new stereogenic centers in one step. Since its discovery, the Diels-Alder reaction has been a cornerstone reaction for the synthesis of organic compounds.
The mechanisms of substituent effects on Diels-Alder reactivity are well understood (30) and the potential for accelerating the reaction by raising the HOMO (highest occupied molecular orbital) energy of the diene, lowering the LUMO (lowest unoccupied molecular orbital) energy of the dienophile, in addition to approximation, provides an attractive target for catalysis. Indeed, several protein catalysts for this reaction have been reported, elicited by immune response against two different transition state analogs (31,32). Experimental studies and quantum mechanical calculations on model systems for the reactions catalyzed indicate that these function by increasing hydrogen-bond strength in the transition state and binding two reactants in an arrangement suitable for reaction (10).
Despite the fact that the Diels-Alder reaction is one of the main chemical routes to make carbon-carbon bonds (e.g., in addition to step-wise aldol condensation), there has been little firm evidence of its use by living organisms as opposed to the many aldolases that have been characterized (33). So far, three natural enzymes have been proposed to catalyze a Diels-Alder reaction, although for some of them the exact mechanism is still debated (34,35,36,37,38,39,40). Consequently, there is no definite evidence of the existence of a natural, bimolecular Diels-Alderase enzyme.
Two different families of synthetic ribozymes (RNA catalysts) have been engineered to date. A library of RNA molecules was created covalently attached to an acyclic diene and selected for Diels-Alder activity (41,42). The best ribozymes showed rate enhancements of up to 800-fold over the uncatalyzed reaction. Since one of the substrates is covalently attached to the RNA catalyst, the reaction is effectively first order and the author reported a kcat/KM of 3.95 M−1s−1 and an effective molarity of 2M. Similar results were obtained using a library of PEG-ylated RNA molecules attached to anthracene and directed to catalyze a Diels-Alder reaction with a maleimide dienophile (43).
A handful of catalytic antibodies have also been elicited for Diels-Alder reactions between various compounds. For example, antibody 1E9 catalyzes the addition of tetrachlorothiophene dioxide and N-ethyl maleimide (44,45). 1E9 is the most effective Diels-Alder catalyzing antibody known to date, with a catalytic proficiency of 107 M−1 and an effective molarity of 103 M (27). In addition, multiple turnovers were observed with antibody 1E9, and a crystal structure was solved showing the molecular details of the active site. Similarly, antibody 39-A11 catalyzes the Diels-Alder reaction between an electron-rich acyclic diene and an N-aryl maleimide (46), although its proficiency is lower due to a less complimentary binding pocket. Finally, antibodies 4D5, 13G5, and several others, were shown to catalyze regio-, diastereo- and enantio-selective addition of 4-carboxybenzyl trans-1,3-butadiene-1-carbamate and N,N-dimethylacrylamide, a model Diels-Alder reaction (10).
The ability to design selective catalysts, including stereoselective catalysts, for the Diels-Alder reaction would be extremely valuable for chemical synthesis. While, an approach for computation enzyme design has been described (Zanghellini et al., New Algorithms and an in silico Benchmark for Computational Enzyme Design, Protein Science 15:2785-2794 (2006)), computational de novo design of an enzyme catalyzing a bimolecular reaction such as a Diels Alder reaction has not been described.